Systemic and local inflammation is a pathological feature observed in many disorders, and in particular metabolic disorders and autoimmune disorders. Low-grade inflammation is an independent risk factor of metabolic diseases and associated co-morbidities such as heart disease, stroke and diabetes. Inflammation predates the detection of insulin resistance and therefore may be a good predictor of diabetes. Chronic (persistent low-grade) and acute (high-grade) inflammation is a key characteristic feature of autoimmune disorders, such as inflammatory bowel disease.
Metabolic syndrome is also known as Syndrome X, metabolic syndrome X, cardiometabolic syndrome, insulin resistance syndrome, CHAOS or Reaven's syndrome. It is generally believed to be a combination of disorders that affect a large number of people in a clustered fashion. The symptoms and features of the syndrome include at least three of the following conditions: diabetes mellitus II, impaired glucose tolerance or insulin resistance, high blood pressure, central obesity and difficulty in losing weight, high cholesterol, combined hyperlipidemia, and fatty liver. On a physiological basis, insulin resistance appears to be responsible for the syndrome. However, insulin resistance can be defined in a myriad of different ways, including impaired glucose metabolism, inability to suppress lipolysis in the tissues, defective protein synthesis, altered cell differentiation, abnormal cell cycle control or proliferation, all of which being implicated in the liver and cardiovascular disease associated with metabolic syndrome.
Although certain bacterial associations have been examined for these conditions, the role of bacterial microbiota in their onset has not been understood yet. And there remains a need for methods for diagnosing, treating, and preventing conditions such as obesity, metabolic syndrome, insulin-deficiency or insulin-resistance related disorders, glucose intolerance, diabetes mellitus, non-alcoholic fatty liver (NAFL), abnormal lipid metabolism, atherosclerosis and related disorders.
Nonalcoholic fatty liver disease NAFLD is the hepatic manifestation of the metabolic syndrome, with insulin resistance as the main pathogenetic mechanism. Non-alcoholic fatty liver disease (NAFLD) represents a spectrum of liver diseases encompassing simple fatty infiltration in liver tissue (steatosis), fat and inflammation of the liver tissue (non-alcoholic steatohepatitis—NASH), and cirrhosis, in the absence of excessive alcohol consumption, viral diseases (HCV, HBV) or other identified etiologies (autoimmune disorders). It is the leading cause of chronic liver diseases in Western countries with a prevalence as high as 30% of the general population. Concerning NASH, studies reported an estimated prevalence of 3 to 5% of the general population. NAFLD is strongly associated with obesity (NAFLD found in more than 90% of obese patients), type 2 diabetes (NAFLD is found in 40% to 70% of T2D patients), and dyslipidaemia. NAFLD may thus be considered as the hepatic manifestation of the metabolic syndrome.
Among the spectrum of NAFLD clinical presentations, simple steatosis has not been associated with liver related morbidity, but NASH was associated with a >10 fold increase risk of liver related death and a doubling of cardiovascular risk. Consequently, NASH is considered as the second most frequent etiology for liver transplant indication, after HCV, representing 18% of patients registered in US transplants list (OPTN).
In fact, both the prevalence and natural history of NAFLD suggest that it is a very common cause of liver disease and that its subtype NASH can progress to cirrhosis. These observations stress out the significant impact of NAFLD in terms of patient health, health-related quality of life and healthcare economics.
Although most patients with NAFLD have steatosis, only a minority progress to more advanced disease, characterized by inflammation and subsequent fibrosis, cirrhosis, and hepatocellular carcinoma. Studies indicate that about 5.4% of patients with NASH develop severe complications of end stage liver disease during long term follow-up. Such progression is probably influenced by genetic and environmental factors, only some of which have been identified. Recognized independent risk factors for progression are age >45 years, presence of diabetes (or severity of insulin resistance), obesity (body mass index >30), and hypertension. The patients who do progress often present late in the natural course of the disease and have substantial liver related morbidity.
There is therefore a need to develop prognosis tests to assess the risk of liver related morbidity for these patients, in particular, to predict the risk of progression from benign fatty liver towards NASH and advanced liver diseases.
Liver biopsy is currently considered as the best tool for assessing degree of severity of the NAFLD, and in particular inflammation and of liver fibrosis. However, liver biopsy is an invasive procedure which is not appropriate or practical outside specialist hepatology practice. Furthermore, it presents drawbacks of interpretation error due to sampling error and to observer variability (Adams L. A. & Feldstein A. E., Journal of Digestive Diseases 2011). There is therefore a clear need to develop reliable non-invasive screening tests to efficiently differentiate the patients who have established NASH versus those who have a steatosis only, as well as patients at low risk of progression from those with more aggressive disease.
NAFLD is often asymptomatic and commonly first discovered as an incidental biochemical abnormality identified during routine blood tests. However, the characteristic biochemical changes (e.g., a relatively greater rise in alanine aminotransferase than in aspartate aminotransferase) tend to reverse, and alanine aminotransferase levels fall as hepatic fibrosis progresses. This means that steatohepatitis with advanced disease may be present even in those with relatively normal alanine aminotransferase levels in blood.
Several other methods have been proposed for non-invasive quantification of hepatic fat and inflammation, including magnetic resonance imaging or spectroscopy and blood biomarker panels. However, evidence supporting their use in wider clinical practice is still limited: routine ultrasound imaging of the liver provides a qualitative assessment of hepatic fat content, but sensitivity is limited, particularly when <33% of hepatocytes are steatotic. And, although they offer greater sensitivity for detecting milder degrees of steatosis, magnetic resonance techniques for lipid quantification are often resource intensive and are not yet widely available for routine clinical use. Furthermore, hepatic fat content tends to diminish as cirrhosis develops, and so NASH is probably consequently under diagnosed in the setting of advanced liver disease.
Hence, no widely accepted, reliable methods are available yet for differentiating simple steatosis from steatohepatitis in routine practice, other than liver biopsy. Consequently, most NAFLD patients remain undiagnosed, and are managed at the primary care level for controlling their underlying metabolic factors (obesity, diabetes, cholesterol . . . ). Patients further progressing towards NASH are identified late in the course of their disease and develop significant liver related morbidity.
There is therefore an urgent need, beyond prognostication, for identification of patients with NAFLD so as to change patient management by (a) providing a greater impetus for modification of diet and lifestyle; (b) guiding drug selection in patients with insulin resistance or diabetes; and (c) allowing specific monitoring strategies to be instituted if cirrhosis is present.
Several therapeutic strategies have been proposed so far to handle the NAFLD patients. However, no drugs are currently approved specifically for treating liver inflammation or fibrosis, the main clinical features of NASH, and many drug candidates failed to demonstrate significant efficacy for treating NASH (reversing established inflammation and fibrosis at histological level). An emerging strategy considers that effective drug treatment should be focused on early onset of NAFLD, to control steatosis and prevent progression to inflammatory stages. This strategy requires effective triage of patients with NAFLD, so that medical care can be tailored to individual's risk of progression towards NASH.
There is therefore also a clear need to develop a screening test to diagnose and clinically differentiate NAFLD suffering patients, for being use as triage test at primary and secondary care level. This test should be additionally non-invasive, and economically acceptable.
The present inventors identified particular gene signatures which are predictive of the evolution of Non Alcoholic Fatty Liver Disease (NAFLD) towards its most severe forms (hepatitis, cirrhosis, liver cancer). These gene signatures are advantageously assessed in stool samples of the patients. These gene signatures are therefore considered to be the first reliable and non-invasive means of diagnosis, prognosis, and stratification for drug studies of liver-related metabolic diseases. They can also be used for monitoring and assigning appropriate treatments to the thus-diagnosed patients.
Autoimmune disorders arise from an inappropriate immune response of the body against substances and tissues normally present in the body. Inflammatory bowel diseases (IBD), such as Crohn's disease or ulcerative colitis, are among the most prevalent autoimmune disorders. These diseases are detected, staged and monitored by 3 main approaches:                Clinical evaluation, mainly using composite scores that integrate both patients data as well as patients self-report questionnaires;        Biological markers, based on blood (i.e. inflammation markers such as CRP, or platelet count), or feces (i.e. calprotectin); and        Imaging tools, including endoscopic exams with or without histologic analysis and magnetic resonance based exams (MRI or MR enterography).        
Any of these approaches are filling the needs for new tools to monitor the disease activity and subsequently the treatment regimen in IBD. The tight control of IBD, thought accurate surveillance and treatment adjustment, is nowadays key in the management of such patients because of the recurring and remitting nature of these disorders.
Monitoring clinical symptoms alone is not reliable enough to assess disease activity. Patients self reporting low disease activity often present intestinal lesions during an endoscopic exam. Biological markers, such as fecal calprotectin, are useful, but non specific and their increase is associated with systemic/mucosal inflammation at the late onset of the flare. Endoscopy enables to detect mucosal healing, which is consider as the most robust and reliable sign of disease remission; however, routine repeated endoscopic monitoring is not feasible, because of the required bowel preparation and general anesthesia. New imaging tools, such as MRI has been shown to be effective, but it is expensive, time-consuming, and limited access precludes routine use. The MR Enterography, presented as the most promising approach, implies also bowel preparation and invasive colonoscopy.
In summary, patients and healthcare providers are actively looking for non-invasive tools enabling evaluation of disease activity and monitoring of patients care.
Here, stable patients are defined as patients for whom disease activity is stable over several weeks (patient in a “stable state”). While instable patients (or patient “in an instable state”) are patients:                who had their treatment changed or intensified in the following weeks,        whose blood tests showed elevated activity in the following weeks, and/or        whose self-evaluation showed decreased health        